Phenolic hydrazone macrophage migration inhibitory factor inhibitors

ABSTRACT

Provided are various compounds of Formula (I): Also provided are pharmaceutical compositions comprising the above compounds. Additionally, methods of inhibiting macrophage migration inhibitory factor (MIF) activity in a mammal are provided, as are methods of treating or preventing inflammation in a mammal. Further provided are methods of treating a mammal having sepsis, septicemia, and/or endotoxic shock. Also provided are methods of treating a mammal having an autoimmune disease, and methods of treating a mammal having a tumor.

CROSS-REFERENCE TO RELATED APPLICATION

This is a U.S. national phase of PCT Application No. PCT/US2007/007277,filed Mar. 23, 2007, which claims the benefit of U.S. ProvisionalApplication No. 60/785,834, filed Mar. 24, 2006.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to cytokine inhibitors. More specifically,the present invention identifies and characterizes several inhibitors ofmacrophage migration inhibitory factor.

(2) Description of the Related Art

Macrophage migration inhibitory factor (MIF) is a potentpro-inflammatory cytokine, critically involved in the pathogenesis ofsepsis and other inflammatory disorders (Calandra and Roger, 2003;Riedemann et al., 2003). Sepsis, a lethal systemic inflammatory reactionto infection, kills more than 215,000 people per annum in the US alone.There is currently no anti-inflammatory therapeutic agent that isapproved by the FDA, for its clinical management. MIF has beendemonstrated to be an important late-acting mediator of systemicinflammation, and inhibiting its activity in vivo attenuates the lethalconsequences of endotoxemia and sepsis in rodents (Calandra et al.,2000; Al-Abed et al., 2005).

MIF exists as a homotrimer (Sugimoto et al., 1995; Sun et al., 1996;Suzuki et al., 1996; Taylor et al., 1999) with the unique ability tocatalyze the tautomerization of non-physiological substrates such asD-dopachrome and L-dopachrome methyl ester into their respective indolederivatives (Rosengren et al., 1996). While the physiological role ofthe tautomerase activity is uncertain, compounds that are structurallysimilar to D- and L-dopachrome can bind to and thereby block the MIF'stautomerase active site (Al-Abed et al., 2005; Cios et al., 2002; Chengand Al-Abed, 2006; Lubetsky et al., 2002; Senter et al., 2002).N-acetyl-p-benzoquinone imine (NAPQI) forms a covalent complex with MIFat its active site (FIG. 1) and is capable of irreversibly inhibitingthe adverse biological effect of MIF (Senter et al., 2002). However, thetoxicity of NAPQI precludes its use as a viable clinical inhibitor ofMIF.

Based on the above, the development of non-toxic small moleculeinhibitors of MIF activity warrants further investigation.

SUMMARY OF THE INVENTION

The inventor has identified and characterized several new compounds thatinhibit MIF activity.

The present invention is thus directed to compounds of Formula I:

where R1 is an alkyl, a substituted alkyl, a cycloalkyl, a substitutedcycloalkyl, a heterocyclic group, a substituted heterocyclic group, anaryl, a substituted aryl, a heteroaryl, a substituted heteroaryl, ahydroxy, an alkoxy, an aryloxy, an oxo, an amino, a halogen, a formyl,an acyl, a carboxy, a carboxyalkyl, a carboxyaryl, an amido, acarbamoyl, a guanidino, a ureido, an amidino, a mercapto, a sulfinyl, asulfonyl or a sulfonamide, and

R2, R3, R4 and R5 are independently a halogen, —OH, —SH, —NH₂, —NO₂,—OR6, or H, where R6 is a straight or branched C₁-C₆ alkyl.

The invention is also directed to pharmaceutical compositions comprisingany of the above compounds, or a pharmaceutically acceptable saltthereof, in a pharmaceutically acceptable carrier.

The present invention is additionally directed to methods of inhibitingmacrophage migration inhibitory factor (MIF) activity in a mammal. Themethods comprise administering the above pharmaceutical composition tothe mammal in an amount effective to inhibit MIF activity in the mammal.

Further, the invention is directed to methods of treating or preventinginflammation in a mammal. The methods comprise administering the abovepharmaceutical composition to the mammal in an amount effective to treator prevent the inflammation in the mammal.

Also, the present invention is directed to methods of treating a mammalhaving sepsis, septicemia, and/or endotoxic shock. The methods compriseadministering the above pharmaceutical composition to the mammal in anamount effective to treat the sepsis, septicemia and/or endotoxic shock.

The invention is further directed to methods of treating a mammal havingan autoimmune disease. The methods comprise administering the abovepharmaceutical composition to the mammal in an amount effective to treatthe autoimmune disease.

Additionally, the present invention is directed to methods of treating amammal having a tumor, the method comprising administering the abovepharmaceutical composition to the mammal in an amount effective to treatthe tumor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a scheme for the rational design of MIFinhibitors.

FIG. 2 is a chemical formula for compound 10, a di-substitutedhydrazone.

FIG. 3 is a graph of experimental results showing that compound 7inhibits TNF secretion from LPS-treated macrophages. RAW 267.4macrophages (10⁵) were treated with various concentrations of compound 7(0.01-100 μM) 30 min prior to LPS addition or 10 μg/ml of mousemonoclonal antibody against MIF (XIV.15.5; α-MIF). After 16 h ofincubation, cell culture supernatants were collected for determinationof TNFα concentration by ELISA. Data are presented as mean±S.D. (n=3, *,p<0.01)

FIG. 4 is a graph of experimental results showing that the hydrazonecompound 7 is protective after 24 h late treatment in a CLP model. Micewere injected intraperitoneally with compound 7 (“hydrazone”) (4 mg/kg)(n=13, p<0.01) or vehicle 24 h after CLP (n=13). A single injection wascomposed of 100 μg of Compound 7 (equivalent to 4 mg/kg) in 200 μL of20% DMSO: 80% saline solution. Additional administrations of compound 7(bi-daily) were given on days 2 and 3.

FIG. 5 is a graph of experimental results showing that compound 7,administered either orally or I.P., inhibits leukocyte recruitment in anestablished model of acute inflammation. *p<0.05 (n=5); ** p<0.006(n=20) relative to vehicle alone.

FIG. 6 is a graph of experimental results showing that compound 7 has anapparent biological half-life of 7-9 hrs as assessed by inhibition ofleukocyte infiltration in an air pouch model challenged withcarrageenan. ** p<0.006 (n=20) relative to vehicle alone.

FIG. 7 is a graph of experimental results showing that compound 7 is aprotective agent in an animal model of endotoxemia.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides several new compounds that inhibit MIFactivity. See Examples.

The present invention is thus directed to compounds of Formula I:

where R1 is an alkyl, a substituted alkyl, a cycloalkyl, a substitutedcycloalkyl, a heterocyclic group, a substituted heterocyclic group, anaryl, a substituted aryl, a heteroaryl, a substituted heteroaryl, ahydroxy, an alkoxy, an aryloxy, an oxo, an amino, a halogen, a formyl,an acyl, a carboxy, a carboxyalkyl, a carboxyaryl, an amido, acarbamoyl, a guanidino, a ureido, an amidino, a mercapto, a sulfinyl, asulfonyl or a sulfonamide, and

R2, R3, R4 and R5 are independently a halogen, —OH, —SH, —NH₂, —NO₂,—OR6, or H, where R6 is a straight or branched C₁-C₆ alkyl.

Preferably, only one of R2, R3, R4 and R5 is not an H. Also preferably,R2 is fluorine or H. More preferably, only R2 is not an H. Even morepreferably, R2 is a halogen and R3, R4 and R5 are all H. Mostpreferably, R2 is fluorine and R3, R4 and R5 are all H.

For any of the above compounds, R1 is preferably COOMe, COOEt, COOtBu,COOCH₂Ph, COOCH₂PhOMe, COPh, SO₂Ph, Me, Ph, PhOMe, COOtBu, Ph, or PhOMe,where Me is CH₃, Ph is a phenyl, and Bu is a butyl. More preferably, R1is COOtBu, Ph, or PhOMe. Even more preferably, the compound is any oneof compounds 4-9 and 11-19 of Tables 1 and 2. Most preferably, thecompound is any one of compounds 5, 6, 7, 8, 9, or 19 of Table 1 or 13,14, 15, or 18 of Table 2. In some aspects, the compound is compound 5 ofTable 1. In other aspects, the compound is compound 6 of Table 1. Instill other aspects, the compound is compound 7 of Table 1. Inadditional aspects, the compound is compound 8 of Table 1. In furtheraspects, the compound is compound 9 of Table 1. Also, the compound canbe compound 19 of Table 1. The compound can also be compound 13 of Table2. Additionally, the compound can be compound 14 of Table 2. Thecompound can further be compound 15 of Table 2. The compound canadditionally be compound 18 of Table 2.

The invention is also directed to pharmaceutical compositions comprisingany of the above compounds, or a pharmaceutically acceptable saltthereof, in a pharmaceutically acceptable carrier.

By “pharmaceutically acceptable” it is meant a material that (i) iscompatible with the other ingredients of the composition withoutrendering the composition unsuitable for its intended purpose, and (ii)is suitable for use with subjects as provided herein without undueadverse side effects (such as toxicity, irritation, and allergicresponse). Side effects are “undue” when their risk outweighs thebenefit provided by the composition. Non-limiting examples ofpharmaceutically acceptable carriers include, without limitation, any ofthe standard pharmaceutical carriers such as phosphate buffered salinesolutions, water, emulsions such as oil/water emulsions, microemulsions,and the like.

The above-described compounds can be formulated without undueexperimentation for administration to a mammal, including humans, asappropriate for the particular application. Additionally, proper dosagesof the compositions can be determined without undue experimentationusing standard dose-response protocols.

Accordingly, the compositions designed for oral, lingual, sublingual,buccal and intrabuccal administration can be made without undueexperimentation by means well known in the art, for example with aninert diluent or with an edible carrier. The compositions may beenclosed in gelatin capsules or compressed into tablets. For the purposeof oral therapeutic administration, the pharmaceutical compositions ofthe present invention may be incorporated with excipients and used inthe form of tablets, troches, capsules, elixirs, suspensions, syrups,wafers, chewing gums and the like.

Tablets, pills, capsules, troches and the like may also contain binders,recipients, disintegrating agent, lubricants, sweetening agents, andflavoring agents. Some examples of binders include microcrystallinecellulose, gum tragacanth or gelatin. Examples of excipients includestarch or lactose. Some examples of disintegrating agents includealginic acid, cornstarch and the like. Examples of lubricants includemagnesium stearate or potassium stearate. An example of a glidant iscolloidal silicon dioxide. Some examples of sweetening agents includesucrose, saccharin and the like. Examples of flavoring agents includepeppermint, methyl salicylate, orange flavoring and the like. Materialsused in preparing these various compositions should be pharmaceuticallypure and nontoxic in the amounts used.

The compounds can easily be administered parenterally such as forexample, by intravenous, intramuscular, intrathecal or subcutaneousinjection. Parenteral administration can be accomplished byincorporating the compounds into a solution or suspension. Suchsolutions or suspensions may also include sterile diluents such as waterfor injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents. Parenteralformulations may also include antibacterial agents such as for example,benzyl alcohol or methyl parabens, antioxidants such as for example,ascorbic acid or sodium bisulfite and chelating agents such as EDTA.Buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose may also beadded. The parenteral preparation can be enclosed in ampules, disposablesyringes or multiple dose vials made of glass or plastic.

Rectal administration includes administering the compound, in apharmaceutical composition, into the rectum or large intestine. This canbe accomplished using suppositories or enemas. Suppository formulationscan easily be made by methods known in the art. For example, suppositoryformulations can be prepared by heating glycerin to about 120° C.,dissolving the composition in the glycerin, mixing the heated glycerinafter which purified water may be added, and pouring the hot mixtureinto a suppository mold.

Transdermal administration includes percutaneous absorption of thecomposition through the skin. Transdermal formulations include patches(such as the well-known nicotine patch), ointments, creams, gels, salvesand the like.

The compounds can also be prepared for nasal administration. As usedherein, nasal administration includes administering the compound to themucous membranes of the nasal passage or nasal cavity of the patient.Pharmaceutical compositions for nasal administration of the compoundinclude therapeutically effective amounts of the compound prepared bywell-known methods to be administered, for example, as a nasal spray,nasal drop, suspension, gel, ointment, cream or powder. Administrationof the compound may also take place using a nasal tampon or nasalsponge.

The compounds of the invention may be administered per se (neat) or inthe form of a pharmaceutically acceptable salt. When used in medicine,the salts should be both pharmacologically and pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare the free active compound or pharmaceuticallyacceptable salts thereof. Pharmacologically and pharmaceuticallyacceptable salts include, but are not limited to, those prepared fromthe following acids: hydrochloric, hydrobromic, sulphuric, nitric,phosphoric, maleic, acetic, salicyclic, p-toluenesulfonic, tartaric,citric, methanesulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzenesulphonic. Also, pharmaceuticallyacceptable salts can be prepared as alkaline metal or alkaline earthsalts, such as sodium, potassium or calcium salts of the carboxylic acidgroup.

The present invention is additionally directed to methods of inhibitingmacrophage migration inhibitory factor (MIF) activity in a mammal. Themethods comprise administering any of the above pharmaceuticalcompositions to the mammal in an amount effective to inhibit MIFactivity in the mammal.

These methods can be used on any mammal. Preferably, the mammal is ahuman. It is also preferred that the mammal has or is at risk for acondition that comprises an inflammatory cytokine cascade that is atleast partially mediated by an MIF. Non-limiting examples of suchconditions include proliferative vascular disease, acute respiratorydistress syndrome, cytokine-mediated toxicity, psoriasis, interleukin-2toxicity, appendicitis, peptic, gastric and duodenal ulcers,peritonitis, pancreatitis, ulcerative, pseudomembranous, acute andischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis,cholecystitis, hepatitis, inflammatory bowel disease, Crohn's disease,enteritis, Whipple's disease, asthma, allergy, anaphylactic shock,immune complex disease, organ ischemia, reperfusion injury, organnecrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia,hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis,septic abortion, epididymitis, vaginitis, prostatitis, urethritis,bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis,alvealitis, bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza,respiratory syncytial virus infection, herpes infection, HIV infection,hepatitis B virus infection, hepatitis C virus infection, disseminatedbacteremia, Dengue fever, candidiasis, malaria, filariasis, amebiasis,hydatid cysts, burns, dermatitis, dermatomyositis, sunburn, urticaria,warts, wheals, vasulitis, angiitis, endocarditis, arteritis,atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardialischemia, periarteritis nodosa, rheumatic fever, Alzheimer's disease,coeliac disease, congestive heart failure, meningitis, encephalitis,multiple sclerosis, cerebral infarction, cerebral embolism,Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury,paralysis, uveitis, arthritides, arthralgias, osteomyelitis, fasciitis,Paget's disease, gout, periodontal disease, rheumatoid arthritis,synovitis, myasthenia gravis, thryoiditis, systemic lupus erythematosus,Goodpasture's syndrome, Behcets's syndrome, allograft rejection,graft-versus-host disease, ankylosing spondylitis, Berger's disease,type 1 diabetes, type 2 diabetes, Berger's disease, Retier's syndromeand Hodgkins disease. A preferred such condition is sepsis, septicemia,and/or endotoxic shock.

MIF has been shown to play an important role in autoimmune disease. See,e.g., Cvetjovic et al., 2005. The present methods would thus be usefulin treatment of autoimmune disease. Thus, in some aspect of thesemethods, the mammal has or is at risk for an autoimmune disease.Non-limiting examples of such autoimmune diseases are multiplesclerosis, systemic lupus erythematosus, rheumatoid arthritis, graftversus host disease, autoimmune pulmonary inflammation, autoimmuneencephalomyelitis, Guillain-Barre syndrome, autoimmune thyroiditis,insulin dependent diabetes mellitus, Crohn's disease, scleroderma,psoriasis, Sjögren's syndrome and autoimmune inflammatory eye disease.

MIF also is known to promote tumor invasion and metastasis. See, e.g.,Sun et al., 2005. The present methods would therefore be useful fortreatment of a mammal that has a tumor.

The invention is also directed to methods of treating or preventinginflammation in a mammal. The methods comprise administering the abovepharmaceutical composition to the mammal in an amount effective to treator prevent the inflammation in the mammal.

For these methods, the mammal is preferably a human. The mammal canhave, or be at risk for, a disease involving inflammation, for exampleproliferative vascular disease, acute respiratory distress syndrome,cytokine-mediated toxicity, psoriasis, interleukin-2 toxicity,appendicitis, peptic, gastric and duodenal ulcers, peritonitis,pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis,diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis,hepatitis, inflammatory bowel disease, Crohn's disease, enteritis,Whipple's disease, asthma, allergy, anaphylactic shock, immune complexdisease, organ ischemia, reperfusion injury, organ necrosis, hay fever,sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia,eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion,epididymitis, vaginitis, prostatitis, urethritis, bronchitis, emphysema,rhinitis, cystic fibrosis, pneumonitis, alvealitis, bronchiolitis,pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytial virusinfection, herpes infection, HIV infection, hepatitis B virus infection,hepatitis C virus infection, disseminated bacteremia, Dengue fever,candidiasis, malaria, filariasis, amebiasis, hydatid cysts, burns,dermatitis, dermatomyositis, sunburn, urticaria, warts, wheals,vasulitis, angiitis, endocarditis, arteritis, atherosclerosis,thrombophlebitis, pericarditis, myocarditis, myocardial ischemia,periarteritis nodosa, rheumatic fever, Alzheimer's disease, coeliacdisease, congestive heart failure, meningitis, encephalitis, multiplesclerosis, cerebral infarction, cerebral embolism, Guillame-Barresyndrome, neuritis, neuralgia, spinal cord injury, paralysis, uveitis,arthritides, arthralgias, osteomyelitis, fasciitis, Paget's disease,gout, periodontal disease, rheumatoid arthritis, synovitis, myastheniagravis, thryoiditis, systemic lupus erythematosus, Goodpasture'ssyndrome, Behcets's syndrome, allograft rejection, graft-versus-hostdisease, ankylosing spondylitis, Berger's disease, type 1 diabetes, type2 diabetes, Berger's disease, Retier's syndrome, or Hodgkins disease.Preferably, the mammal has sepsis, septicemia, and/or endotoxic shock,or is at risk for sepsis, septicemia, and/or endotoxic shock.

These methods can include the administration of a secondanti-inflammatory agent to the mammal. Examples of such secondanti-inflammatory agents are NSAIDs, salicylates, COX inhibitors, COX-2inhibitors, and steroids. Preferably, the mammal has or is at risk forsepsis, septicemia, and/or endotoxic shock and the second treatment isadministration of a muscarinic agonist, an adrenomedullin, anadrenomedullin binding protein, a milk fat globule epidermal growthfactor VIII, an activated protein C, or an α2A-adrenergic antagonist.

The present invention is also directed to methods of treating a mammalhaving sepsis, septicemia, and/or endotoxic shock. The methods compriseadministering the above pharmaceutical composition to the mammal in anamount effective to treat the sepsis, septicemia and/or endotoxic shock.

The invention is further directed to methods of treating a mammal havingan autoimmune disease. The methods comprise administering the abovepharmaceutical composition to the mammal in an amount effective to treatthe autoimmune disease. Examples of such autoimmune diseases includemultiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis,graft versus host disease, autoimmune pulmonary inflammation, autoimmuneencephalomyelitis, Guillain-Barre syndrome, autoimmune thyroiditis,insulin dependent diabetes mellitus, Crohn's disease, scleroderma,psoriasis, Sjögren's syndrome and autoimmune inflammatory eye disease.

Additionally, the present invention is directed to methods of treating amammal having a tumor, the method comprising administering the abovepharmaceutical composition to the mammal in an amount effective to treatthe tumor.

As established in Example 2 below, these compounds can be effectivelyadministered orally. Thus, in any of the above methods, thepharmaceutical composition can be administered orally. Alternatively,the pharmaceutical composition can be administered parenterally.

Preferred embodiments of the invention are described in the followingexample. Other embodiments within the scope of the claims herein will beapparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims, which follow the examples.

EXAMPLE 1 Phenolic Hydrazones are Potent Inhibitors of MacrophageMigration Inhibitory Factor Proinflammatory Activity and areSurvival-Improving Agents in Sepsis Example Summary

A series of phenolic hydrazones were synthesized and evaluated for theirinhibition of MIF activity. Compound 7 emerged as a potent inhibitor ofMIF tautomerase with an IC₅₀ of 130 nM. Compound 7 dose-dependentlysuppressed TNFα secretion from lipopolysaccharide-stimulatedmacrophages. The therapeutic importance of the MIF inhibition bycompound 7 is demonstrated by the significant protection from thelethality of sepsis when administration of the compound was initiated ina clinically relevant time frame.

Introduction

A rational design approach was used to produce a more potent, smallmolecule inhibitor of MIF. The indole intermediate of MIF tautomerasecatalysis presented itself as a suitable template for the development ofpotential MIF inhibitors. It was reasoned that compounds designed aroundthe phenyl imine scaffold could act as potential MIF antagonists.Indeed, from this rational design the amino acid Schiff bases and theisoxazoline compounds as MIF tautomerase inhibitors were developed (Dioset al., 2002; Lubetsky et al., 2002). Among the amino acid Schiff basestested for their ability to inhibit the tautomerase activity of MIF, itwas found that compound 1 was the most potent (FIG. 1, Table 1).Recently, an isoxazoline inhibitor of MIF, “ISO-1” (compound 2, FIG. 1)was reported, which blocks the tautomerase site, inhibits the ability ofMIF to overcome anti-inflammatory glucocorticoid activities in vitro andimproves survival in animal models of experimental sepsis (Lubetsky etal., 2002).

To find more potent inhibitors of MIF, the phenyl imine scaffold wasrevisited and modified by adding nitrogen to afford hydrazone-typecompounds (FIG. 1). Previous studies on the amino acid Schiff bases andcompound 2 revealed that a para-hydroxyl group, as exemplified by aphenolic moiety, is a key structural feature and is required foractivity. Replacing the phenolic moieties of 1 and 2 with either phenyl-or halide-substituted phenyl or p-methoxyphenyl groups resulted in thedecrease or complete loss of their ability to inhibit MIF tautomeraseactivity (Dios et al., 2002; Lubetsky et al., 2002). In accordance withthis result, the chemical structure of the new pharmacophores mustcontain a phenolic ring (FIG. 1). Utilizing this key structural featureand the observations of 1 and 2 complexed with MIF, phenolic hydrazones3-19 (Tables 1 and 2) were synthesized as potential MIF inhibitors.

TABLE 1 Phenolic hydrazones and IC₅₀ values for inhibition of MIFtautomerase activity.

Compounds^(a) Ar R IC₅₀ (μM)^(b)  1 1.6 L-tryptophan Schiff base  2 7ISO-1  3

H >500  4

CH₃ 43  5

2.6  6^(c)

0.48  7^(c)

0.13  8^(c)

0.22  9^(c)

0.33 16

COPh 36.5 17

SO₂Ph 86 19

1.5 ^(a)Compounds were characterized by ¹H, ¹³C NMR and MS.^(b)Spectrophotometric analysis of MIF tautomerase activity onL-dopachrome methyl ester (see experimental). ^(c)See experimental forgeneral procedure for the synthesis of 6-9.

TABLE 2 Phenolic hydrazone carbamates and IC₅₀ values for inhibition ofMIF tautomerase activity.

Compounds^(a) Ar R′ IC₅₀ (μM)^(b) 11

CH₃ 55 12

CH₂CH₃ 43 13

C(CH₃)₃ 5.5 14

10 15

8 18

C(CH₃)₃ 2.4 ^(a)Compounds were characterized by ¹H, ¹³C NMR and MS (seesupporting information). ^(b)Spetrophotometric analysis of MIFtautomerase activity on L-dopachrome methyl ester.

All of the hydrazones prepared in this study, compounds 3-19, have onecommon key structural feature, in that they all possess a “phenolichead” in the form of a 4-hydroxyphenyl ring. It has been shown that thephenolic ring forms key hydrogen bond interactions between the aminoacid residue asparagine-97C of the hydrophobic surface within the MIFactive site (Dios et al., 2002; Lubetsky et al., 2002; Orita et al.,2001). In addition to this important hydrogen bond interaction, there isa hydrophobic interaction that exists between the aromatic ring of thephenol and the side chains of the amino acid residues, Pro-1, Met-2,Ile-64, Tyr-95, Val-106 and Phe-113, of the hydrophobic pocket, thatfurther contributes to the binding of the inhibitor (Orita et al.,2001). Supporting evidence for the key interaction between the hydroxylfunctionality and the amino acid residue asparagine-97C was obtainedby 1) modifying its position and 2) replacing the hydroxyl group withother functional groups. The position of the hydroxyl group is acritical feature of the hydrazones in that changing its position frompara (IC₅₀ 2.5 μM) to meta (IC₅₀ 150 μM) resulted in a dramatic loss ofits ability to inhibit MIF tautomerase activity. Furthermore, replacingthe hydroxyl group with hydrogen, fluoro, amino, methoxy and nitrofunctionalities afforded hydrazones that were inactive (data not shown).In developing a structure-activity relationship between the phenolichydrazones and MIF, hydrazones 3-6 were synthesized from the simplehydrazine, methyl hydrazine, phenyl hydrazine and p-methoxyphenylhydrazine. One further prerequisite for activity of our phenolichydrazones is that they require a hydrophobic tail. This is evident fromcompound 3 (IC₅₀=>500 μM). The hydrogen substituent of the hydrazone inthis compound does not offer any hydrophobic interactions and as aresult is a very poor inhibitor of MIF tautomerase activity (Table 1).Upon replacing the hydrogen with a methyl group, a pronouncedimprovement in the ability of the methyl hydrazone 4 to inhibit thetautomerase activity of MIF (IC₅₀=43 μM) was observed. This suggeststhat there is a hydrophobic interaction between the methyl group and thesurface of MIF. Replacing the methyl group of 4 with the morehydrophobic phenyl ring affords the hydrazone 5 (IC₅₀=2.6 μM) that is 16times more potent. Installation of a para-methoxy group on the aromaticring of 5, gives hydrazone 6 that is 5-fold more potent as an inhibitorthan the parent compound. This increased binding of the p-methoxy phenylhydrazone may be explained by hydrogen bond interactions between theether oxygen and the known amino acid residues, and the possibility ofpi-pi stacking and/or van der Waals interactions between the p-methoxyphenyl ring and the second hydrophobic region of the MIF active site. Ithas been reported that the amino acids, Pro-33, Tyr-36, Phe-49, Trp-108,and Phe-113, make up the second hydrophobic surface at the rim of theactive site of MIF (Dios et al., 2002; Orita et al., 2001). Theseresidues further contribute to the hydrophobic and hydrogen bondinteractions between the pharmacophore and the active site of MIF (Oritaet al., 2001). In further support of these interactions, it waspreviously shown that the L-amino acid Schiff bases inhibitory effectwas improved by five-fold upon changing the amino acid residue fromL-phenylalanine (IC₅₀=50 μM) to L-tyrosine (IC₅₀=10 μM), respectively.This suggests that the increased potency was attributed to a hydrogenbond interaction within residues at the rim of MIF (Dios et al., 2002).Hydrazone 6 is twelve times more potent an inhibitor than L-tryptophanSchiff base 1, so far the most potent inhibitor of MIF described in theliterature (Al-Abed et al., 2005; Dios et al., 2002; Cheng and Al-Abed,2006; Orita et al., 2001; Orita et al., 2002). Recently, it wasdiscovered that mono-fluorination of 2 improved the inhibition of MIFactivity (Cheng and Al-Abed, 2006). As a consequence, we synthesized the3-fluoro, 3-chloro, and 3-bromo-4-hydroxyphenyl derivatives of the morepotent hydrazone, namely the p-methoxyphenyl hydrazone (6). Thesynthesis of the mono-halogenated hydrazone derivatives involvedtreatment of a suspension of the 4-methoxyphenylhydrazine hydrochlorideand the 3-halogenated-4-hydroxybenzaldehyde in methanol with aqueoussodium hydroxide (Table 1). A general increase in the inhibition of theMIF tautomerase activity was observed for the3-halogenated-4-hydroxyphenyl hydrazone derivatives 7-9 (Table 1). Amongthese hydrazones, compound 7 showed the most potent inhibition with anIC₅₀ value of 130 nM, whilst 8 and 9 gave values of 220 nM and 330 nM,respectively. The significant improvement in the inhibitory effect ofthese halogenated hydrazones 7-9 may be explained by the inductiveeffect that may lead to changes in the polarization of the hydroxylmoiety thereby making it a stronger hydrogen bond donor/acceptor.

To determine if the activity would be affected by disubstitution on thenitrogen, compound 10 was synthesized (FIG. 2). Methylation of phenylhydrazine (5) with methyl iodide and sodium amide affordedN-methyl-N-phenyl hydrazine. Hydrazone formation under typicalconditions gave 10 in 86% yield. Compound 10 was a very weak inhibitorof MIF activity, displaying an IC₅₀ value of 300 μM. This observationcould be accounted for by increased steric hinderance or by the reducednumber of hydrogen bonding interactions between the di-substitutedhydrazone derivative 10 and the active site of MIF.

To further investigate the structure-activity relationship between thehydrazones and MIF another class of compounds, the phenolic hydrazonecarbamates, were evaluated. The phenolic hydrazone carbamates 11-15 and18 were chosen as they displayed key functionalities similar to those ofthe amino acid Schiff base 1 and 2 (Table 2). These functionalities arethe hydroxyl group, phenyl ring and the carboxylate moiety. It has beenreported that a secondary hydrogen bond interaction exists between thecarboxylate moiety of 2 and lysine-32A of the MIF active site (Dios etal., 2002). Improvement in the inhibition of MIF tautomerase activity ofapproximately 10-fold was observed on changing the methyl group of 11(IC₅₀=55 μM) to the more lipophilic t-butyl moiety 13 (IC₅₀=5.5 μM). Todetermine if phenyl rings improve the inhibition of MIF activity, thet-butyl carbamate was replaced with a benzyl carbamate 14 and p-methoxybenzyl carbamate 15. This change from a bulky alkyl 13 to an aromaticgroup 14-15 (IC₅₀=10, 8 μM) resulted in a slight decrease in theinhibitory effect.

Biological Activity. Intracellular MIF occupies a critical role inmediating the cellular responses to pathways activated bylipopolysaccharide (LPS) endotoxin (Roger et al., 2001) andMIF-deficient cells are hyporesponsive to endotoxin (Bozza et al.,1999). MIF null macrophages can produce 50-60% less TNF compared to wildtype (Al-Abed et al., 2005; Mitchell et al., 2002). Additionally,anti-MIF antibody (10 μg/ml) inhibits 50% of TNF release fromLPS-treated macrophages (Al-Abed et al., 2005). Compound 2 (ISO-1)dose-dependently inhibits LPS-induced TNF release from wild-type but notMIF null macrophages, suggesting that the chemical inhibitor of MIF isspecific. Accordingly, it was reasoned that compound 7 binding to MIFwould suppress LPS responses in macrophages. Compound 7 dose-dependentlydid inhibit LPS-induced TNF release (FIG. 3). Thus, compound 7recapitulates the phenotype of the MIF deficient macrophages and isassociated with decreased TNF production in response to LPS.

The importance of MIF as a molecular therapeutic target in sepsis hasbeen confirmed by the recent observation that treatment with anti-MIFantibodies or compound 2 significantly improves survival in septic mice(Al-Abed et al., 2005). Further, serum MIF levels increased to 70% ofmaximum levels within 24 h post CLP, and peaked at 36 h (Al-Abed et al.,2005). This identified MIF as a late mediator in sepsis and indicatedthe therapeutic potential of inhibiting MIF in a clinically relevanttime frame. Therefore, it was reasoned that a delayed treatment withcompound 7, consistent with the kinetics of MIF release, could besuccessfully applied to improve survival in sepsis. The ability ofcompound 7 to improve the survival rate in cecal ligation and puncture(CLP)-induced peritonitis, a widely used preclinical model of sepsis,was tested. Intraperitoneal treatment of 7 (4 mg/kg) initiated 24 hafter CLP surgery and continued for 3 days resulted in a survival rateof 65% (p<0.01) compared to 28% in the control (vehicle-treated) group(FIG. 4). Thus, compound 7 treatment provides significant protectionagainst sepsis lethality, comparable to the effect of anti-MIF antibodyand compound 2 (Al-Abed et al., 2005). Remarkably, a dose of compound 7,10-fold less than 2, achieved similar protection. This finding indicatesan association between the potency of compound 7 in inhibiting the MIFtautomerase active site and its beneficial effect of improving survivalin experimental sepsis.

In summary, phenolic hydrazones were designed and synthesized as nontoxic, potent MIF antagonists. The structure-activity relationship studysuggests that minor changes in functionalization of the hydrazonesaffect the binding of these compounds to the MIF tautomerase activesite. Notably, compound 7 exhibits the greatest activity of all thecompounds tested and is so far the most potent inhibitor of MIFdescribed in the literature (Al-Abed et al., 2005; Dios et al., 2002;Cheng and Al-Abed, 2006; Orita et al., 2001; Orita et al., 2002).

Compound 7 exhibited a potent anti-inflammatory activity in vitro asdemonstrated by suppression of LPS-induced macrophage activation (FIG.3). Moreover, a relatively low concentration of this small molecule MIFinhibitor improved survival in sepsis when treatment was initiated at 24hours after the onset of the disease.

Sepsis is a complex inflammatory disorder and its clinical management isa challenging health issue. Therefore, the finding that compound 7 iseffective at 24 hours after the onset of the disorder could be ofconsiderable clinical interest. Additionally, anti-cytokine agents thatshow efficacy in rodent sepsis models have proved to be valuabletherapeutics for a variety of human inflammatory and autoimmune diseasesand conditions, such as rheumatoid arthritis and Crohn's disease.

Experimental Section

General Experimental. All chemicals were obtained from commercialsuppliers and used without further purification. Aluminium-backed SilicaGel 60 with a 254 nm fluorescent indicator TLC plates were used.Developed TLC plates were visualized under a short-wave UV lamp, stainedwith an I₂—SiO₂ mixture. Flash column chromatography (FCC) was performedusing flash silica gel (32-63 μm) and usually employed a stepwisesolvent polarity gradient, correlated with TLC mobility. Melting points(M.p.) were determined in a Gallenkamp Melting Point Apparatus in opencapillaries and are uncorrected. IR spectra were obtained on a ThermoNicolet IR 100 FT-IR spectrometer. All ¹H spectra were recorded eitheron a Jeol spectrometer or a GE QE 300 spectrometer at 270 or 300 MHz.The ¹³C spectra were recorded on a GE QE 300 spectrometer at 75 MHz.Chemical shifts are relative to the deuterated solvent peak and are inparts per million (ppm). The coupling constants (J) are measured inHertz (Hz). The ¹H signals are described as s (singlet), d (doublet), t(triplet), q (quartet), m (multipet) and br s (broad singlet). Low andhigh resolution mass spectroscopy was carried out at the MassSpectrometry Facility at the University of Illinois at Urbana-Champaign.

General Experimental for compounds 3-5 and 10-19. 4-Hydroxybenzaldehyde(122 mg, 1 mmol) or 3-fluoro-4-hydroxybenzaldehyde (140 mg, 1 mmol) andthe hydrazide (2 mmol) were dissolved in ethanol (10 mL). To this wasadded acetic acid (1 mmol) and the reaction was stirred overnight atroom temperature. Removal of the ethanol in vacuo afforded an oilyresidue. The residue was taken up with ethyl acetate and washed withwater. The organic layer was separated and dried with anhydrous Na₂SO₄.Concentration in vacuo afforded a residue which was subsequentlypurified by FCC using hexane and ethyl acetate as eluent (4:1) to givecompounds 3-5 and 10-15.

General Procedure for compounds 6-9. 4-Hydroxybenzaldehyde (122 mg, 1mmol) or 3-fluoro-4-hydroxybenzaldehyde (140 mg, 1 mmol) or3-chloro-4-hydroxydroxybenzaldehyde (156 mg, 1 mmol) or3-bromo-4-hydroxybenzaldehyde (201 mg, 1 mmol) and4-methoxyphenylhydrazine hydrochloride (350 mg, 2 mmol) were suspendedin methanol (10 mL). To this suspension was added a 2M aqueous solutionof sodium hydroxide (60 mg, 1.5 mmol) and the reaction was stirredovernight at room temperature. Upon completion of the reaction, thesolution was then acidified to pH 4 by the addition of 1M HCl. Removalof the methanol in vacuo afforded an oily residue. The residue was takenup with ethyl acetate and washed with water. The organic layer wasseparated and dried with anhydrous Na₂SO₄. Concentration in vacuoafforded a residue which was subsequently purified by FCC using hexaneand ethyl acetate as eluent (4:1) to give compounds 6-9.

Spectrophotometric Assay for Enzymatic Activity. A fresh stock solutionof L-dopachrome methyl ester (2.4 nM) was generated by oxidation ofL-3,4-dihydroxyphenylalanine methyl ester with sodium periodate,producing an orange-colored solution. Activity was determined at roomtemperature by adding dopachrome methyl ester (0.3 mL) to a cuvettecontaining 1 μL of MIF solution (850 ng/mL) in 50 mM potassium phosphatebuffer, pH 6, and measuring the decrease in absorbance from 2 to 20 s at475 nm spectrophotometrically. The inhibitors 3-15 were dissolved inDMSO at various concentrations (0.1-100 μM) and 1 μL was added to thecuvette with the MIF prior to the addition of the dopachrome.

Cellular Assay. Compound 7 inhibits TNF secretion from LPS-treatedmacrophages. RAW 267.4 macrophages (10⁵) were treated with variousconcentrations of hydrazone 7 (0.01-100 μM) 30 min prior to LPSaddition. After 16 h of incubation, cell culture supernatants werecollected for determination of TNF concentration by ELISA. Data arepresented as mean±S.D. (n=3, *, p<0.1).

Animal studies. All animal experiments were approved by theInstitutional Animal Care and Use Committee of the North Shore-LongIsland Jewish Research Institute. Male Balb/C mice, ˜8 weeks old, weresubjected to cecal ligation and puncture. Details of the CLP procedurehas been carried out as follows: In anesthetized male Balb/C mice(ketamine 100 mg/kg and xylazine 8 mg/kg administered intramuscularly)the cecum was ligated and given a single puncture. Abdominal access wasgained via a midline incision. The cecum was isolated and ligated with a6-0 silk ligature below the ileocecal valve, and the cecum puncturedonce with a 22 G needle, stool (approximately 1 mm) extruded from thehole, and the cecum placed back into the abdominal cavity. The abdomenwas closed with two layers of 6-0 Ethilon sutures. Antibiotics wereadministered immediately after CLP (Premaxin 0.5 mg/kg, subcutaneously,in a total volume of 0.5 ml/mouse) and single dose of resuscitativefluid (normal saline solution administered subcutaneously (20 ml/kg-bodyweight) immediately after CLP surgery (Wang et al., 1999). Mice wereinjected intraperitoneally with 4 mg/kg (n=13, **P<0.01) or vehicle 24hours after CLP (n=13). Additional two injections were given on day 2and 3. Vehicle (aqueous 20% DMSO) or compound 7 (4 mg/kg;intraperitoneally) treatment was started 24 h after the induction ofsepsis and repeated twice daily on days 2 and 3. Animal survival wasmonitored for 14 days.

NMR Data

Compound 3: Yellow solid (90%). M.p. 248-250° C. IR (nujol mull) ν cm⁻¹:3220, 3315, 1650, 1603, 1568, 1506. ¹H NMR (300 MHz, CD₃OD) δ 6.99 (d,2H, J=8.8 Hz, H3, H5), 7.94 (d, 2H, J=8.8 Hz, H2, H6), 8.87 (s, 1H, H7).¹³C NMR (75 MHz, CD₃OD) δ 117.9 (C3, C5), 121.7 (C1), 134.7 (C4, C6),163.2 (C7), 166.1 (C4). MS (ESI) 137.1 (M+1, 25). ESIHRMS m/z calcd forC₇H₉N₂O 137.0715, found 137.0719.

Compound 4: Red solid (85%). M.p. 58-60° C. IR (nujol mull) ν cm⁻¹:3361, 1671, 1604, 1513. ¹H NMR (300 MHz, (CD₃)₂CO) δ2.81 (d, 3H, 3.3 Hz,Me), 6.87 (d, 2H, J=8.8 Hz, H3, H5), 7.42 (d, 2H, J=8.8 Hz, H2, H6),7.95 (s, 1H, H7), 8.35 (br s, 2H, OH, NH, D₂O exchangeable). ¹³C NMR (75MHz, (CD₃)₂CO) δ 36.1 (Me), 116.5 (C3, C5), 127.9 (C1), 129.6 (C2, C6),151.2 (C7), 157.8 (C4). MS (ESI) 151.1 (M+1, 100), 152.1 (M+2, 8).ESIHRMS m/z calcd for C₈H₁₁N₂O 151.0871, found 151.0874.

Compound 5: Brown solid (88%). M.p. 154-156° C. IR (nujol mull) ν cm⁻¹:3406, 3291, 1599, 1508. ¹H NMR (300 MHz, (CD₃)₂CO) δ 6.70 (dd, 1H,J=7.5, 1.2 Hz, H4′), 6.82 (d, 2H, J=8.4 Hz, H3, H5), 7.07 (dd, 2H,J=7.5, 1.2 Hz, H2′, H6′), 7.16 (t, 2H, J=7.5 Hz, H3′, H5′), 7.50 (d, 2H,J=8.4 Hz, H2, H6), 7.77 (s, 1H, H7), 8.50 (br s, 1H, D₂O exchangeable),9.15 (s, 1H, D₂O exchangeable). ¹³C NMR (75 MHz, (CD₃)₂CO) δ 113.1 (C2′,C6′), 116.4 (C3, C5), 119.5 (C4′), 128.2 (C3′, C5′), 128.8 (C1), 129.8(C2, C6), 138.2 (C7), 146.9 (C1′), 158.6 (C4). MS (ESI) 213.1 (M+1, 85),214.1 (M+2, 10). ESIHRMS m/z calcd for C₁₃H₁₃N₂O 213.1028, found213.1030.

Compound 6: Brown residue (84%). IR (nujol mull) ν cm⁻¹: 3419, 1681,1670, 1650, 1635, 1603, 1507. ¹H NMR (300 MHz, (CD₃)₂CO) δ 3.65 (s, 3H,OMe), 6.78 (d, 2H, J=8.7 Hz, H3′, H5′), 6.80 (d, 2H, J=8.4 Hz, H3, H5),7.01 (d, 2H, J=8.7 Hz, H2′, H6′), 7.47 (d, 2H, J=8.4 Hz, H2, H6), 7.72(s, 1H, H7), 8.40 (br s, 1H, D₂O exchangeable), 8.91 (s, 1H, D₂Oexchangeable). ¹³C NMR (75 MHz, (CD₃)₂CO) δ 55.9 (OMe), 114.7 (C3′,C5′), 115.4 (C2′, C6′), 116.6 (C3, C5), 128.4 (C1), 130.2 (C2, C6),137.2 (C7), 141.2 (C1′), 151.2 (C4′), 163.9 (C4). MS (ESI) 241.1 (M−1,65). ESIHRMS m/z calcd for C₁₄H₁₃N₂O₂ 241.0977, found 241.0981.

Compound 7: Brown solid (89%). M.p. 120-121° C. IR (nujol mull) ν cm⁻¹:3319, 3302, 1619, 1514. ¹H NMR (300 MHz, (CD₃)₂CO) δ 3.69 (s, 3H, OMe),6.80 (d, 2H, J=8.8 Hz, H3′, H5′), 6.95 (t, 1H, J=8.8 Hz, H5), 7.04 (d,2H, J=8.8 Hz, H2′, H6′), 7.21 (d, 1H, J=8.4 Hz, H6), 7.42 (dd, 1H,J=10.6, 1.8 Hz, H2), 7.70 (s, 1H, H7), 8.78 (br s, 1H, D₂Oexchangeable), 9.11 (s, 1H, D₂O exchangeable). ¹³C NMR (75 MHz,(CD₃)₂CO) δ 56.0 (OMe), 113.3 (J_(CCF)=19.5 Hz, C2), 114.3 (C3′, C5′),115.5 (C2′, C6′), 118.7 (C5), 123.4 (C6), 130.2 (J_(CCCF)=4.9 Hz, C1),135.9 (C7), 140.7 (C1′), 145.6 (J_(CCF)=13.7 Hz, C4), 152.6(J_(CF)=239.2 Hz, C3), 154.4 (C4′). MS (ESI) 261.1 (M+1, 30), 262.1(M+2, 6). ESIHRMS m/z calcd for C₁₄H₁₄N₂O₂F 261.1039, found 261.1048.

Compound 8: Brown solid (89%). M.p. 108-110° C. IR (nujol mull) ν cm⁻¹:1601, 1557, 1501. ¹H NMR (300 MHz, (CD₃)₂CO) δ 3.69 (s, 3H, OMe), 6.80(d, 2H, J=8.8 Hz, H3′, H5′), 7.03 (d, 2H, J=8.8 Hz, H2′, H6′), 7.13 (d,1H, J=8.4 Hz, H5), 7.32 (dd, 1H, J=6.6, 1.8 Hz, H6), 7.54 (d, 1H. J=1.8Hz, H2), 7.67 (s, 1H, H7), 8.80 (br s, 1H, D₂O exchangeable), 9.12 (s,1H, D₂O exchangeable). ¹³C NMR (75 MHz, (CD₃)₂CO) δ 55.9 (OMe), 114.3(C3′, C5′), 115.5 (C2′, C6′), 117.8 (C5), 121.6 (C3), 126.4 (C6), 127.6(C2), 130.6 (C1), 135.5 (C7), 140.6 (C1′), 153.5 (C4), 154.4 (C4′). MS(ESI) 277.1 (MCl³⁵+1, 60), 279.1 (MCl³⁷+1, 25). ESIHRMS m/z calcd forC₁₄H₁₃N₂O₂Cl 277.0744, found 277.0739.

Compound 9: Brown solid (90%). M.p. 135-137° C. IR (nujol mull) ν cm⁻¹:3453, 3305, 1597. ¹H NMR (300 MHz, (CD₃)₂CO) δ 3.69 (s, 3H, OMe), 6.80(d, 2H, J=8.8 Hz, H3′, H5′), 6.97 (d, 1H, J=8.4 Hz, H5), 7.03 (d, 2H,J=8.8 Hz, H2′, H6′), 7.45 (dd, 1H, J=6.2, 2.2 Hz, H6), 7.69 (s, 1H, H7),7.78 (d, 1H, J=1.8 Hz, H2), 8.85 (s, 1H, D₂O exchangeable), 9.15 (s, 1H,D₂O exchangeable). ¹³C NMR (75 MHz, (CD₃)₂CO) δ 55.9 (OMe), 110.8 (C3),114.2 (C3′, C5′), 115.5 (C2′, C6′), 117.4 (C5), 127.1 (C6), 130.8 (C2),131.0 (C1), 135.3 (C7), 140.6 (C1′), 154.4 (C4′), 154.5 (C4). MS (ESI)321.0 (MBr⁷⁹+1, 100), 323.0 (MBr⁸¹+1, 80). ESIHRMS m/z calcd forC₁₄H₁₃N₂O₂Br 321.0239, found 321.0240.

Compound 10: White solid (86%). M.p. 93-95° C. IR (nujol mull) ν cm⁻¹:3300, 1600, 1506. ¹H NMR (300 MHz, (CD₃)₂CO) δ 3.38 (s, 3H, Me), 6.81(t, 1H, J=8.4 Hz, H4′), 6.83 (d, 2H, J=8.8 Hz, H3, H5), 7.24 (t, 2H,J=8.4 Hz, H3′, H5′), 7.37 (dd, 2H, J=8.1, 1.1 Hz, H2′, H6′), 7.57 (d,2H, J=8.8 Hz, H2, H6), 7.60 (s, 1H, H7), 8.43 (s, 1H, OH, D₂Oexchangeable). ¹³C NMR (75 MHz, (CD₃)₂CO) δ 33.1 (Me), 115.5 (C2′, C6′),116.3 (C3, C5), 120.5 (C4′), 128.3 (C3′, C5′), 129.6 (C2, C6), 129.8(C1), 133.5 (C7), 149.2 (C1′), 158.3 (C4). MS (ESI) 227.1 (M+1, 100),228.1 (M+2, 20). ESIHRMS m/z calcd for C₁₄H₁₅N₂O 227.1184, found227.1174.

Compound 11: White solid (90%). M.p. 165-167° C. IR (nujol mull) ν cm⁻¹:3253, 1651, 1635, 1606, 1556, 1514. ¹H NMR (300 MHz, (CD₃)₂CO) δ 33.78(s, 3H, OMe), 6.93 (d, 2H, J=8.8 Hz, H3, H5), 7.60 (d, 2H, J=8.8 Hz, H2,H6), 8.08 (s, 1H, H7), 8.78 (s, 1H, D₂O exchangeable), 9.85 (br s, 1H,D₂O exchangeable) ¹³C NMR (75 MHz, (CD₃)₂CO) δ 52.4 (OMe), 116.4 (C3,C5), 127.4 (C1), 129.3 (C2, C6), 144.9 (C7), 154.9 (C4), 159.8 (C═O). MS(ESI) 195.1 (M+1, 100), 196.1 (M+2, 10). ESIHRMS m/z calcd for C₉H₁₁N₂O₃195.0770, found 195.0776.

Compound 12: White solid (89%). M.p. 189-190° C. IR (nujol mull) ν cm⁻¹:3338, 3202, 1678, 1607, 1581, 1558, 1513. ¹H NMR (300 MHz, (CD₃)₂CO) δ1.21 (t, 3H, J=7.0 Hz, OCH₂CH₃), 4.13 (q, 2H, J=7.0 Hz, OCH₂CH₃), 6.84(d, 2H, J=8.8 Hz, H3, H5), 7.50 (d, 2H, J=8.8 Hz, H2, H6), 8.00 (s, 1H,H7), 8.73 (br s, 1H, D₂O exchangeable), 9.74 (br s, 41, D₂Oexchangeable). ¹³C NMR (75 MHz, (CD₃)₂CO) δ 15.1 (OCH₂CH₃), 61.5(OCH₂CH₃), 116.4 (C3, C5), 127.5 (C₁), 129.3 (C2, C6), 144.8 (C7), 154.4(C4), 159.8 (C═O). MS (ESI) 209.1 (M+1, 100), 210.1 (M+2, 10). ESIHRMSm/z calcd for C₁₀H₁₃N₂O₃ 209.0926, found 209.0932.

Compound 13: White solid (89%). M.p. 155-156° C. IR (nujol mull) □νcm⁻¹: 3346, 3243, 1660, 1608, 1578, 1534, 1510. ¹H NMR (300 MHz, (CD₃)₂δCO) δ 1.48 (s, 9H, C(CH₃)₃), 6.87 (d, 2H, J=8.8 Hz, H3, H5), 7.53 (d,2H, J=8.8 Hz, H2, H6), 8.01 (s, 1H, H7), 8.69 (br s, 1H, D₂Oexchangeable), 9.58 (br s, 1H, D₂O exchangeable). ¹³C NMR (75 MHz,(CD₃)₂CO) δ 28.7 (C(CH₃)₃), 80.2 (C(CH₃)₃), 116.4 (C3, C5), 127.7 (C1),129.2 (C2, C6), 144.2 (C7), 153.4 (C4), 159.6 (C═O). MS (ESI) 237.1(M+1, 30), 238.1 (M+2, 5). ESIHRMS m/z calcd for Cl₁₂H₁₇N₂O₃ 237.1239,found 237.1249.

Compound 14: White solid (90%). M.p. 159-160° C. IR (nujol mull) ν cm⁻¹:3397, 1668, 1606, 1556, 1506. ¹H NMR (300 MHz, (CD₃)₂CO) δ 5.16 (s, 2H,OCH₂), 6.84 (d, 2H, J=8.4 Hz, H3, H5), 7.26-7.35 (m, 3H, ArCH), 7.36 (d,2H, J=8.8 Hz, ArCH) 7.51 (d, 2H, J=8.4 Hz, H2, H6), 8.00 (s, 1H, H7),8.71 (s, 1H, D₂O exchangeable), 9.91 (br s, 1H, D₂O exchangeable). ¹³CNMR (75 MHz, (CD₃)₂CO) δ 67.1 (OCH₂Ar), 116.4 (C3, C5), 127.4 (C1),128.8 (CH), 129.2 (C2, C6), 129.3 (CH), 137.9 (CCH), 145.2 (C7), 154.3(C4), 159.8 (C═O). MS (ESI) 271.1 (M+1, 100), 272.1 (M+2, 10). ESIHRMSm/z calcd for C₁₅H₁₅N₂O₃ 271.1083, found 271.1089.

Compound 15: White solid (89%). M.p. 147-148° C. IR (nujol mull) ν cm⁻¹:3412, 3270, 1683, 1602, 1543, 1514. ¹H NMR (300 MHz, (CD₃)₂CO) δ 3.76(s, 3H, OMe), 5.08 (s, 2H, OCH₂), 6.83 (d, 2H, J=8.4 Hz, H3, H5), 6.89(d, 2H, J=8.8 Hz, CHCOMe), 7.33 (d, 2H, J=8.4 Hz, CHCCOMe), 7.51 (d, 2H,J=8.8 Hz, H2, H6), 8.00 (s, 1H, H7), 8.70 (s, 1H, D₂O exchangeable),9.82 (br s, 1H, D₂0 exchangeable). ¹³C NMR (75 MHz, (CD₃)₂CO) δ 55.6(OMe), 66.9 (OCH₂), 114.6 (C3, C5), 116.4 (CCOMe), 127.4 (C1), 129.3(C2, C6), 129.8 (CCCCOMe), 130.7 (CCCOMe), 145.0 (C7), 154.3 (C4), 159.8(C═O), 160.6 (COMe). MS (ESI) 301.1 (M+1, 25), 302.1 (M+2, 5). ESIHRMSm/z calcd for C₁₆H₁₇N₂O₄ 301.1188, found 301.1200.

Compound 16: white solid (86%). ¹H NMR (300 MHz, (CD₃)₂CO) ν 3.76 (s,3H), 5.08 (s, 2H), 6.82 (d, J=8.4 Hz, 2H), 6.87 (d, J=8.8 Hz, 2H), 7.31(d, J=8.4 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.99 (s, 1H), 8.70 (s, 1H).MS: m/z 299.5 (M-H).

Compound 17: white solid (87%). ¹H NMR (270 MHz, (CD₃)₂CO) δ 6.89 (d,J=7.9 Hz, 2H), 7.54 (m, 5H), 7.94 (d, J=7.9 Hz, 2H), 8.40 (s, 1H), 8.85(s, 1H). MS: m/z 239.3 (M-H).

Compound 18: white solid (89%). ¹H NMR (300 MHz, (CD₃)₂CO) δ 1.44 (s,9H), 6.98 (dd, J=2.5, 8.4, 1H), 7.27 (d, J=8.0 Hz, 1H), 7.41 (dd, J=2.2,12.1 Hz, 1H), 7.97 (s, 1H), 8.98 (s, 1H). MS: m/z 253.3 (M-H).

Compound 19: brown solid (87%). ¹H NMR (270 MHz, (CD₃)₂CO) δ 6.77 (t,J=6.9 Hz, 1H), 7.01 (t, J=8.4 Hz, 1H), 7.12-7.29 (m, 4H), 7.48 (dd,J=1.7, 10.4 Hz, 1H), 7.77 (s, 1H). MS: m/z 229.7 (M-H).

Spectrophotometric Assay for Enzymatic Activity. A fresh stock solutionof L-dopachrome methyl ester (2.4 nM) was generated by oxidation ofL-3,4-dihydroxyphenylalanine methyl ester with sodium periodate,producing an orange-colored solution. Activity was determined at roomtemperature by adding dopachrome methyl ester (0.3 mL) to a cuvettecontaining 1 μL of MIF solution (850 ng/mL) in 50 mM potassium phosphatebuffer, pH 6, and measuring the decrease in absorbance from 2 to 20 s at475 nm spectrophotometrically. The inhibitors were dissolved in DMSO atvarious concentrations (0.1-100 mM) and added to the cuvette with theMIF prior to the addition of the dopachrome.

Animal studies. Compound 7 is protective even when treatment isinitiated after 24 h in a CLP model (FIG. 4). Mice were injectedintraperitoneally with 3.5 mg/kg (n=13, **P<0.01) or vehicle 24 hoursafter CLP (n=13). Two additional injections were given on day 2 and 3.Details of the CLP procedure has been carried out as follows: Inanesthetized male Balb/C mice (ketamine 100 mg/kg and xylazine 8 mg/kgadministered intramuscularly). Abdominal access was gained via a midlineincision. The cecum was isolated and ligated with a 6-0 silk ligaturebelow the ileocecal valve, and the cecum punctured once with a 22Gneedle, stool (approximately 1 mm) extruded from the hole, and the cecumreplaced in the abdominal cavity. The abdomen was closed with two layersof 6-0 Ethilon sutures. Antibiotics were administered immediately afterCLP (Premaxin 0.5 mg/kg, subcutaneously, in a total volume of 0.5ml/mouse) and single dose of resuscitative fluid (normal saline solutionadministered subcutaneously (20 ml/kg-body weight) immediately after CLPsurgery (Wang et al., 1999).

EXAMPLE 2 Effect of Compound 7 on Leukocyte Recruitment in Response toAcute Inflammation

Air pouches were made according to standard procedures (Garcia-Ramalloet al., 2002) on Swiss Webster male mice (25-30 g) by injecting sterileair s.c. on day 0 (6 ml) and day 3 (3 ml). On day 6, animals weretreated with vehicle (350 μl of 20% DMSO) or compound 7 (7 mg/kg) eitherintraperitoneal (i.p.) or gavage (oral) as indicated. After 15 min, theanimals were challenged by injecting 1 ml 1% carrageenan (in PBS) intothe air pouch cavity. Five hrs after carrageenan injection the animalswere sacrificed, the pouches washed with PBS, exudate collected, and thetotal number of infiltrating cells quantitated. The plot shows thenumber of cells normalized to that seen with vehicle alone (veh.).

Results are shown in FIG. 5. Both the i.p. and oral treatments withcompound 7 showed a significant reduction in leukocyte recruitment inresponse to the inflammation caused by the carrageenan treatment intothe air pouch cavity.

In another experiment, air pouches were made as above. On day 6, animalswere treated with vehicle (350 μl of 20% DMSO) or compound 7 (7 mg/kg,gavage) at the indicated times prior to sacrificing and cell harvesting.In all cases, 1% carrageenan was injected into the air pouch cavity 5hrs before sacrificing.

Results are shown in FIG. 6. The plot shows the number of cellsnormalized to that seen with vehicle alone (veh.). When the mice weretreated orally with compound 7 at 5.25 and 7 hours, but not 9 hours,before harvesting air pouch cells for quantitation, a significantreduction in leukocyte recruitment was observed. This indicates thatwith oral administration, compound 7 has an apparent biologicalhalf-life of 7-9 hours.

EXAMPLE 3 Compound 7 Protects Mice from Fatal Endotoxemia

Endotoxemia was induced in Balb/C mice by injection of LPS (16 mg/kg,top or 19 mg/kg, bottom). Mice were treated with either vehicle (350 μl20% DMSO) or compound 7 (7 mg/kg) i.p., 2 hrs before and 24 after LPSinfusion. Survival of the mice was monitored.

Results are shown in FIG. 7. Compound 7 afforded protection to the micefrom otherwise fatal LPS exposure.

REFERENCES

-   Al-Abed, Y.; Dabideen, D.; Aljabari, B.; Valster, A.; Messmer, D. et    al. ISO-1 binding to the tautomerase active site of MIF inhibits its    pro-inflammatory activity and increases survival in severse    sepsis. J. Biol. Chem. 2005, 280, 36541-35644.-   Bozza, M.; Satoskar, A. R.; Lin, G.; Lu, B.; Humbles, A. A. et al.    Targeted disruption of migration inhibitory factor gene reveals its    critical role in sepsis. J. Exp. Med. 1999, 189, 341-346.-   Calandra, T.; Echtenacher, B.; Roy, D. L.; Pugin, J.; Metz; C. N.;    Hultner, L.; Heumann, D.; Mannel, D.; Bucala, R.; Glauser, M. P.    Protection from septic shock by neutralization of macrophage    migration inhibitory factor. Nat. Med. 2000, 6, 164-170.-   Calandra, T.; Roger, T. Macrophage migration inhibitory factor: a    regulator of innate immunity. Nat. Rev. Immunol 2003, 3, 791-800.-   Cvetkovic, I.; Al-Abed, Y. et al. Critical role of macrophage    migration inhibitory factor activity in experimental autoimmune    diabetes. Endocrinol. 2005, 146, 2942-2951.-   Cheng, K. F.; Al-Abed, Y. Critical modifications of the ISO-1    scaffold improve its potent inhibition of macrophage migration    inhibitory factor (MIF) tautomerase activity. Bioorg. Med. Chem.    Lett. 2006, 16, 3376-9.-   Dios, A.; Mitchell, R. A.; Aljabari, B.; Lubetsky, J.; O'Connor, K.    A.; Liao, H.; Senter, P. D.; Manogue, K. R.; Lolis, E.; Metz, C.;    Bucala, R.; Callaway, D. J. E.; Al-Abed, Y. Inhibition of MIF    bioactivity by rational design of pharmacological inhibitors of MIF    tautomerase activity. J. Med. Chem. 2002, 45, 2410-2416.-   Garcia-Ramallo E.; Marques, T. et al. Resident cell chemokine    expression serves as the major mechanism for leukocyte recruitment    during local inflammation. J. Immunol. 2002, 169, 6467-6473-   Lubetsky, J. B.; Dios, A.; Han, J.; Aljabari, B.; Ruzsicska, B.;    Mitchell, R.; Lolis, E.; Al-Abed, Y. The tautomerase active site of    macrophage migration inhibitory factor is a potential target for    discovery of novel anti-inflammatory agents. J. Biol. Chem. 2002,    277, 24976-24982.-   Mitchell, R. A.; Liao, H.; Chesney, J.; Fingerle-Rowson, G.;    Baugh, J. et al. Macrophage migration inhibitory factor (MIF)    sustains macrophage proinflammatory function by inhibiting p53:    regulatory role in the innate immune response. Proc Natl Acad Sci    USA 2002, 99, 345-350-   Orita, M.; Yamamoto, S.; Katayama, N.; Aoki, M.; Takayama, K.;    Yamagiwa, Y.; Seki, N.; Suzuki, H.; Kurihara, H.; Sakashita, H.;    Takeuchi, M.; Fujita, S.; Yamada, T.; Tanaka, A. Coumarin and    chromen-4-one analogues as tautomerase inhibitors of macrophage    migration inhibitory factor: Discovery and X-ray crystallography. J.    Med. Chem. 2001, 44, 540-547.-   Orita, M.; Yamamoto, S.; Katayama, N.; Fujita, S. Macrophage    migration inhibitory factor and the discovery of tautomerase    inhibitors. Curr. Pharm. Des. 2002, 8, 1297-1317.-   Riedemann, N. C.; Guo, R. F.; Ward, P. A. Novel strategies for the    treatment of sepsis. Nat. Med. 2003, 9, 517-524.-   Roger, T.; David, J.; Glauser, M. P.; Calandra, T. MIF regulates    innate immune responses through modulation of Toll-like receptor 4.    Nature 2001, 414, 920-924.-   Rosengren, E.; Bucala, R.; Aman, P.; Jacobsson, L.; Odh, G. et al.    The immunoregulatory mediator macrophage migration inhibitory factor    (MIF) catalyzes a tautomerization reaction. Mol. Med. 1996, 2,    143-149.-   Senter, P. D.; Al-Abed, Y.; Metz, C. N.; Benigni, F.;    Mitchell, R. A. et al. Inhibition of macrophage migration inhibitory    factor (MIF) tautomerase and biological activities by acetaminophen    metabolites. Proc Natl Acad Sci USA 2002, 99, 144-149-   Sugimoto, H.; Suzuki, M.; Nakagawa, A.; Tanaka, I.; Fujinaga, M. et    al. Crystallization of rat liver macrophage migration inhibitory    factor for MAD analysis. J. Struct. Biol. 1995, 115, 331-334.-   Sun, B.; Nishihira, J. et al. Macrophage migration inhibitory factor    promotes tumor invasion and metastasis via the Rho-dependent    pathway. Clin. Cancer Res. 2005, 11, 1050-1058.-   Sun, H. W.; Bernhagen, J.; Bucala, R.; Lolis, E. Crystal structure    at 2.6-A resolution of human macrophage migration inhibitory factor.    Proc. Natl. Acad. Sci. U S A 1996, 93, 5191-5196.-   Suzuki, M.; Sugimoto, H.; Nakagawa, A.; Tanaka, I.; Nishihira, J. et    al. Crystal structure of the macrophage migration inhibitory factor    from rat liver. Nat. Strict. Biol. 1996, 3, 259-266.-   Taylor, A. B.; Johnson, W. H., Jr.; Czerwinski, R. M.; Li, H. S.;    Hackert, M. L. et al. Crystal structure of macrophage migration    inhibitory factor complexed with (E)-2-fluoro-p-hydroxycinnamate at    1.8 A resolution: implications for enzymatic catalysis and    inhibition. Biochemistry 1999, 38, 7444-7452.-   Wang, H.; Bloom, O.; Zhang, M.; Vishnubhakat, J. M.; Ombrellino, M.    et al. HMG-1 as a late mediator of endotoxin lethality in mice.    Science 1999, 285, 248-251

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

1. A method of treating an inflammatory condition in a mammal, whereinthe inflammatory condition is sepsis, septicemia, endotoxemia orrheumatoid arthritis, comprising administering to the mammal an amountof the compound of Formula I effective to treat an inflammatorycondition in a mammal, wherein the compound of Formula I is:

wherein R1 is COOtBu, COOCH₂Ph, COOCH₂PhOMe, Ph or PhOMe, where Me isCH₃, Ph is a phenyl and Bu is a butyl, and R2, R3, R4 and R5 areindependently a halogen or H, wherein either (i) only one of R2, R3, R4and R5 is not H, or (ii) R2, R3, R4 and R5 are H.
 2. The method of claim1, wherein only one of R2, R3, R4 and R5 is not an H.
 3. The method ofclaim 2, wherein R2 is a halogen.
 4. The method of claim 2, wherein R2is a fluoro group.
 5. The method of claim 1, wherein R1 is PhOMe.
 6. Themethod of claim 5, wherein R2 is a fluoro group, and R3, R4 and R5 areH.
 7. The method of claim 5, wherein R2 is a chloro group, and R3, R4and R5 are H.
 8. The method of claim 5, wherein R2 is a bromo group, andR3, R4 and R5 are H.
 9. The method of claim 5, wherein R2, R3, R4 and R5are H.
 10. The method of claim 1, wherein R1 is COOtBu.
 11. The methodof claim 10, wherein R2 is a fluoro group.
 12. The method of claim 10,wherein R2, R3, R4 and R5 are H.
 13. The method of claim 1, wherein R1is Ph.
 14. The method of claim 13, wherein R2 is a fluoro group, and R3,R4 and R5 are H.
 15. The method of claim 13, wherein R2, R3, R4 and R5are H.
 16. The method of claim 1, wherein R2, R3, R4 and R5 are H. 17.The method of claim 1, wherein the inflammatory condition is sepsis. 18.The method of claim 1, wherein the inflammatory condition is septicemia.19. The method of claim 1, wherein the inflammatory condition isendotoxemia.
 20. The method of claim 1, wherein the inflammatorycondition is rheumatoid arthritis.